Amino acids in the cloned mouse kappa receptor that are necessary for high affinity agonist binding but not antagonist binding

Abstract

Opioids exert their biological effects through membrane-bound receptors. We have been interested in examining the domains and specific residues of the opioid receptors involved in ligand binding. In this report we demonstrate the critical roles of two highly conserved aspartate residues in the second and third transmembrane domains (TM2 and TM3) of the κ receptor in ligand binding. Two mutant receptors were generated in which the negatively charged aspartate residues residing in TM2 and TM3 were independently mutated to neutrally charged asparagines (D105N and D138N). These aspartate residues are absolutely required for high affinity κ-selective agonist binding. In addition, we exchanged the region encompassing TM4 and the second extracellular loop between the κ and δ opioid receptors. One of these chimeras, the κ receptor with TM4 and the δ second extracellular loop (κ/δ-2eloop), was unable to bind κ-selective agonists but bound antagonists and nonselective opioids in a manner identical to wild-type κ receptor. The ability of κ-selective agonists to inhibit forskolin-stimulated cAMP accumulation in COS-7 cells transfected with the κ/δ-2eloop chimera was completely abolished, consistent with the loss of binding observed. These results indicate that TM4 and/or the second extracellular loop are involved in κ-selective agonist binding.